Adenosine dextran conjugates

ABSTRACT

Covalent conjugates of water soluble dextrans and adenosine agonists or antagonists wherein the dextran is coupled through the C6 or C8 positions of the purine ring. These compounds activate or block adenosine A 1  or A 2  receptors are useful in treating hypertension.

This application is a continuation of application Ser. No. 07,874,044,filed on Apr. 27, 1992, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to adenosine agonist and antagonistcompounds covalently coupled to medium molecular weight and highmolecular weight dextrans and microbeads. These adenosine agonist andantagonist complexes alone or in combination are able to selectivelyactivate intravascular endothelial adenosine receptors as well asextravascular adenosine receptors.

2. Discussion of the Background

Adenosine is a low molecular weight (Mw=267) naturally occurringnucleoside having several receptor-mediated effects in mammals withpotential therapeutic use. These effects of adenosine can be blocked atthe receptor level by theophylline and other methylxanthines. Theeffects of intravascular administration of adenosine include: coronaryand general vascular dilation, inhibition of the release of renin andcatecholamine, auricular-ventricular blockade and spontaneousventricular tachycardial depression and reduction of myocardialperfusion injury. Thus, adenosine can potentially be a coronaryvasodilator, an anti-hypertensive agent (by causing general vasculardilation and inhibition of the release of renin and catecholamines) andan antiarrhythmic agent and a protective agent against myocardialinfarction.

However, when adenosine is given intravascularly, it distributes itselfthroughout the entire extracellular (intravascular plus intrastitial)compartment, thereby being able to affect all cells containing adenosinereceptors. Additionally, adenosine is rapidly taken up by all cells andinactivated by metabolizing enzymes. These two properties limit thetherapeutic use of this nucleoside. Due to lack of compartmentalization,adenosine cannot act solely on specific target cells, but affects allcells and thereby loses specificity. Further, adenosine is rapidlymetabolized so that its effects are very transitory and have nostability.

The cardiovascular actions of adenosine have been extensively studiedand include coronary vasodilation, a negative chronotropic/dromotropiceffect an anti-adrenergic response and cardiac protection againstinfarction (Olafsson et al, Circulation, 76(5):1135-1145 (1987); Babbittet al, Circulation, 80:1388-1399 (1989); Liu et al, Circulation,84:350-356 (1991)). While it is now known that most of these actions aremediated via membrane bound receptors, the precise mechanisms by whichadenosine exerts its cardiovascular effects have yet to be defined.Difficulties arise secondary to the numerous influences involved withthe formation and metabolism of adenosine. With the myocardium,adenosine can be produced and metabolized by both the endothelium andcardiomyocytes, and is therefore subject to the influence of both celltypes. The relative contribution of these two cell types to theextracellular level of adenosine is dependent on several physiologicalfactors and is still an area of controversy. There is also uncertaintyas to the actual site of action of adenosine and whether or not some ofits vascular effects are in part mediated via the vascular endothelium.

Endothelial dependent vascular relaxation is a well studied phenomenaand has been documented for several substances including acetylcholine,ATP, ADP and substance P. For a review see Furchgott, Circ. Res.,53:557-73, 1983 and Luscher et al, CRC, Boca Raton, pp. 1-87, 1990.However, it has been difficult to establish the role of the vascularendothelium with respect to adenosine mediated vascular relaxation. Forinstance, studies on isolated arterial segments have shown a reductionin the vasodilatory effects of adenosine in endothelial denuded arterialsegments. See Frank, G. W. and Bevan, J. A., Regulatory Function ofAdenosine, Berne, R. M., Rall, T. W., Rubio, R. (eds), Martinus Nijhoff,Hague Boston London, p. 511 (1983); Haendrick, J., Berne, R. M., Am. J.Physiol., 259:H62-H67 (1990); Kennedy, C., Burnstock, G., Blood Vessels,22:145-155 (1985); Moritoki, H., Role of Adenosine and AdenineNucleotides in the Biological System, Imai S., Nakazawa, M. (eds),Elseveir, Amsterdam New York Oxford, pp. 217-224 (1991); Rubanyi, G.Vanhoutte, P. M., J. Cardiovasc. Pharmacol., 7:139-144 (1985). At thesame time, adenosine vasodilatory effects have been reported to beindependent of the vascular endothelium. See Cassis, L. A., Loeb, A. L.,Peach, M. J., Topics and Perspectives in Adenosine Research, Gerlach,E., Becker, B. F. (eds), Springer, Berlin Heidelberg New York, pp.486-496 (1987); Luscher, T. F., Vanhoutte, P.M., CRC, Boca Raton, pp.1-87 (1990); Mathieson, J. I., Burnstock, G., Europ. J. Pharmacol.,118:221-229 (1985); Pearson, J. D., Gordon, J. L., Nature, 181:384-186(1979). In some intact vascular beds, the endothelium may be necessaryfor the maximum vasodilatory response to adenosine. Nonetheless, it isnot possible to assess the relative contribution of the vascularendothelium using conventional methods of comparing blood vesselresponses both with and without the endothelium. This is because thevascular endothelium of intact vascular beds cannot be denuded withoutaltering organ function. Moreover, the relative importance of theendothelium becomes evident if one considers that adenosine remainsconfined to the intravascular compartment during its intracoronaryinfusion, in up to micromolar concentrations, secondary to theimpermeable metabolic barrier imposed by the endothelium (see Nees, S.,Herzog, V., Becker, B. F., Bock, M., Des Rosiers, C., Gerlach, E., BasicRes. Cardiol., 80:515-529 (1985); Nees, S., Herzog, V., Becker, B. F.,Bock, M., Des Rosiers, C., Gerlach, E., Adenosine: Receptors andModulation of Cell Function, Stefanovich, V., Rudolph, K., Schubert, P.(eds), IRI, Oxford, pp. 419-436 (1985); Nees, S., Gerlach, E.,Regulatory Function of Adenosine, Berne, R. M., Rall, T. W., Rubio, R.(eds), Martinus Nijhoff, Hague Boston London, pp. 347-360 (1983)) andyet the vasodilatory and negative dromotropic effects of adenosine areobservable at these concentrations (Nees, S., Gerlach, E., ibid. ).

The pharmacokinetics of macromolecular adenosine analogs are similar totheir low molecular weight counterparts during intracoronary infusion.Nees et al have studied the metabolic effects of perfusing isolatedguinea-pig hearts with polyadenylic acid (poly-A; molecular weight:100,000). See Nees, S., Herzog, V., Becker, B. F., Bock, M., DesRosiers,, C., Gerlach, E., Basic. Res. Cardiol., 80:515-529 (1985).Olsson et al covalently bonded adenosine and theophylline to oxidizedstachyose. Anesthetized dogs were then administered these compounds byintracoronary infusion to study dose-dependent coronary vasodilation.See Olsson, R. A., Davis, C. J., Khouri, E. M., Patterson, R. E., Cir.Res., 39:93-98 (1976). Schrader et al covalently coupled adenosinemonophosphate (AMP) to the enzyme carbonic anhydrase to produce aconjugate having a mean molecular weight of about 30,000. When infusedinto the coronary arteries of isolated guinea-pig hearts, the conjugateinduced vasodilation which was similar in magnitude and time course tothe vasodilation elicited by free AMP or adenosine. See Schrader, J.,Nees, S., Gerlach, E., Pfluger Arch., 369:251-257 (1977). Intracoronaryinfusion of adenosine deaminase, which deaminates adenosine to inosine,alters cardiovascular function and yet this enzyme remains largelyintravascularly confined. Clemo, H. F., Belardinelli, L., Cir. Res.,59:437-446 (1986).

Although Schrader et al, Nees et al and Olsson et al couple adenosine tolarger molecules, these derivatives are not large enough to completelyprevent the passage of the derivatives through the endothelium andoutside of the vascular compartment. Selective activation ofintravascular adenosine receptors is not possible with these relativelylow molecular weight adenosine derivatives.

There is a continuing need for adenosine agonist and antagonistcompounds which are useful in studying the functional significance ofintravascular, endogenous and exogenous coronary adenosine. Further,adenosine compounds which selectively activate intravascular orinterstitial adenosine receptors or which block these receptors areuseful pharmaceutical agents in eliciting specific cardiovasculareffects in mammals.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide adenosineagonist and antagonist compounds which are useful in studyingcardiovascular adenosine receptors.

Another object of the invention is to provide adenosine agonist andantagonist pharmaceutical compounds which selectively bind tointravascular adenosine receptors.

A further object is to provide adenosine agonist and antagonistcompounds which alone or in combination can be tailored to elicit adesired specific cardiovascular response.

These and other objects which will become apparent from the followingspecification have been achieved by the present adenosine agonist andantagonist compounds which are covalently coupled to medium molecularweight and high molecular weight dextrans or microbeads. Whenadministered intravascularly, the medium molecular weight compoundsequilibrate between the extracellular and the intravascularcompartments. In contrast, the high molecular weight dextran compoundsand compounds coupled to microbeads remain exclusively in theintravascular compartment. When a combination of high molecular weightdextran-adenosine antagonist and medium molecular weightdextran-adenosine agonist are administered intravascularly, the agonistwill act solely on extravascular receptors because the intravasculareffects are prevented by the high molecular weight dextran-antagonist.These selective distributions allow one to control the cardiovascularresponse to administration of the adenosine agonist and antagonist byadjusting the molecular weight of the coupled compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of preferred adenosine derivativesof the present invention covalently bonded to a polymer microbead;

FIGS. 2(A) and 2(B) show the decrease in coronary perfusion pressure andventricular developed pressure induced by adenosine agonist derivatives(NOA and ADAC); FIG. 2(C) shows a decrease in the maximal frequency ofatrial pacing to producing a 1:1 A-V propagation induced by microbeadcoupled NOA or microbead coupled ADAC, and the blockade of themicrobead-coupled NOA effect by SPT; and FIG. 2(D) shows the decrease inglycolytic flux and coronary perfusing pressure caused by sustainedinfusion of microbead-coupled NOA;

FIG. 3(A) and FIG. 3(B) show the negative dromotropic effect caused byintracoronary infusion of microbead-coupled NOA and the blockade of thiseffect by SPT, A-V delay (ordinates) at different frequencies of atrialpacing (abscissas);

FIG. 4(A) and FIG. 4(B) show the negative dromotropic effect caused byintracoronary infusion of microbead-coupled ADAC, A-V delay (ordinates)at different frequencies of atrial pacing (abscissas);

FIG. 5 shows a selective blockade of the hypoxia-induced lengthening inA-V delay by intracoronary infusion of microbead-coupled XAC;

FIG. 6 shows the effect of 10 μl adenosine boluses (10⁻³ M, shown byarrow) on mean coronary perfusion pressure at a constant coronary flowof 10 ml/min. FIGS. 6(A-14 C) show pressure readings from individualexperiments indicating a drop in perfusion pressure A: control duringK-H only perfusion. B,C: bolus given after 5 minute of infusion withSPH-XAC and with XAC 10⁻⁷ M, respectively. FIGS. 6(D-E) show compileddata for adenosine bolus experiments. D: control A (n=22) during K-Honly perfusion, control B (n=7) during control microbead infusion, thenduring SPH-XAC (n=11), XAC 10 ⁻⁵ M (n=6) and XAC 10⁻⁷ M (n=6) infusion.E: effect of decreasing SPH-XAC concentration from 6.0 to 0,006 mg/ml(n=3);

FIG. 7 shows the time progression of Mobitz type II nodal heart blockduring 10 μl bolus injections of adenosine (10⁻³ M) at a stimulationfrequency of 3.5 Hz.FIG. 7(A) : effect of SPH-XAC (6.0 mg/ml, n=6)infusion. FIG. 7(B): effect of XAC 10⁻⁷ M (n=6) infusion. Controls inFIG. 7(A-B) are K-H perfusion only (n=7);

FIG. 8 shows the effect of hypoxia (95% N₂ +5% CO₂) on the A-V intervaland recovery. FIG. 8(A): control (n=8) and during 6.0 mg/ml SPH-XAC(n=7) infusion. FIG. 8(B): comparison during infusion of XAC 10⁻⁷ M(n=4) and during infusion of 6.0 mg/ml SPH-XAC;

FIG. 9 shows the effect of hypoxia on coronary pressure. FIGS. 9(A-C):individual pressure readings during a 2.0 minute period of hypoxia (95%N₂ +5% O₂). A: control, K-H perfusion only. B: 6.0 mg/ml SPH-XACinfusion. C: 10⁻⁷ M free XAC infusion. FIG. 9(D): drop in mean coronarypressure during hypoxia (95% N₂ +5% O₂) for control (n=10), XAC 10⁻⁷ M(n=4), SPH-XAC 6.0 mg/ml (n=8).FIG. 9(E): drop in mean coronary pressureduring hypoxia (75% N₂ +20% O₂ +5% CO₂) for control (n=5), XAC 10⁻⁷ M(n=4), SPH-XAC 6.0 mg/ml (n=4);

FIG. 10 shows the results of an individual experiment in whichintracoronary perfusion of dextran-coupled ADAC produced a decrease incoronary perfusion pressure and in ventricular developed pressure; and

FIG. 11 shows the results of sustained intracoronary infusion ofdextran-coupled ADAC on A-V delay;

FIG. 12 shows that upon perfusion with a hypoxic medium, A-V delaylengthens and upon reoxygenation, this effect is reversed (control).However, if the same insult is applied during a sustained infusion ofdextran-XAC (0.1 mg/ml), the hypoxic effect is substantially blocked.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, the nucleoside adenosine, an adenosine agonistor an adenosine antagonist are covalently coupled to a microbead or to adextran. Since these compounds are larger than adenosine, they are notaccessible to intracellular adenosine metabolizing enzymes and are,therefore, more stable, remain in the circulation and act for longerperiods of time than adenosine. By manipulating the molecular size ofthe adenosine derivative, one gains specificity by regulating thedistribution of the adenosine compound between the intravascular andextravascular/interstitial compartments, thereby preferentiallyaffecting a specific adenosine receptor population.

The high molecular weight and medium molecular weight adenosine agonistsof the present invention are substitutes for adenosine in pharmaceuticaland research applications. The agonists of the present inventioninteract with the adenosine A₁ and A₂ receptors producing physiologicaleffects similar to adenosine itself. However, the agonists of thepresent invention have significantly prolonged effects since thesehigher molecular weight agonists are not enzymatically degraded byintracellular adenosine metabolizing enzymes.

The high molecular weight and medium molecular weight: antagonists ofthe present invention are useful in pharmaceutical applicationsrequiring blockade of adenosine A₁ receptors, for examplehypoxia-related conditions. Hypoxia (low blood oxygen partial pressure)frequently occurs in coronary artery disease and pulmonary disease.Hypoxia results in the production of endogenous adenosine whichincreases the A-V delay. Blockade of the endothelial A₁ adenosinereceptors with the antagonists of the present invention significantlyshortens the A-V interval, increasing heart rate thereby providingincreased oxygen to the patient.

The effects of a high molecular weight adenosine agonist or antagonistcompound result only from endothelial adenosine receptor activation orblockade since the high molecular weight adenosine agonist or antagonistcompound is retained within the intravascular compartment. Conversely, amedium molecular weight adenosine agonist or antagonist compound can acton endothelial as well as extravascular cells, since the mediummolecular weight compounds can pass through the vascular wall.

As used herein, the term "adenosine derivative" refers to adenosine, alladenosine agonists and adenosine antagonists which may be covalentlybonded or coupled to a polymer microbead or to a dextran having amolecular weight of about 1,000-5,000,000 Daltons. The term "highmolecular weight" refers to an adenosine derivative covalently bonded toa polymer microbead or to an adenosine derivative covalently bonded to adextran having a molecular weight of about 1,000,000-5,000,000 Daltons(1,000-5,000 kD). The term "medium molecular weight" refers to anadenosine derivative covalently bonded to a dextran having a molecularweight in the range of about 1,000 up to 1,000,000 Daltons (1-999 kD).The terms "medium molecular weight" and "high molecular weight" are usedherein to qualitatively describe the vascular distribution properties ofadenosine derivatives coupled to microbeads or dextrans. Obviously,adenosine derivatives coupled to dextrans having molecular weight justbelow 1,000,000 Daltons (1,000 kD) will exhibit properties similar tothe high molecular weight coupled compounds.

A diversity of polymer microbeads made by polymerization orcopolymerization of a diversity of monomers including, but not limitedto styrene, divinylbenzene, maleic anhydride, acrylamide, etc., arecommercially available having a particle size range from about 0.01microns to about 100 microns average diameter (Bangs, Seradyn, Inc.).These microbeads are manufactured using known methods and arecharacterized by a diversity of surface functional groups such as:carboxylic (--COOH), amidic (--CO--NH₂), aldehydic (--CHO), aromaticamine (--C₆ H₅ --NH₂), hydrazidic (--NH--NH₂) and hydroxylic (--OH)groups. These surface functional groups are used to covalently bond theadenosine derivative to the microbead. The chemical reactions which areused to form covalent bonds between the surface functional groups of themicrobead and the adenosine derivative are well known in the art. Forexample, carboxylate modified microbeads may be covalently bonded to anadenosine derivative through amide or ester linkages. Hydroxyl surfacegroups on the microbead can be reacted using appropriate chemicalreactions to form ether, ester or carbamate linkages. Aromatic aminesurface functional groups can be used to form amide, carbamate orurethane linkages. The diversity of surface functional groups permitsthe selection of known chemistries in coupling the adenosine derivativesto the microbeads. Preferred microbeads are carboxylate modified polymermicrobeads having an average diameter greater than 0.015 microns.

Particularly preferred carboxylate modified polymer microbeads arecommercially available having a surface charge density ranging fromabout 0.1 to about 0.3 meq/g and average diameters of about 0.05-1.0microns (Bangs; Seradyn Inc.). Preferred carboxylate modified microbeadsare produced by copolymerizing a vinyl carboxylic acid or anhydride withstyrene to produce a carboxylate modified polystyrene latex microbead.

An adenosine derivative may be covalently coupled to microbeads orderivatized, i.e., carboxylate modified, microbeads by any knowncoupling reaction. The method of coupling the adenosine derivative tothe microbead is not critical, so long as the covalently coupled productretains activity and is stable under physiological conditions. That is,the adenosine derivative can be covalently bonded to the microbead byany known method which produces a product in which the adenosinederivative does not dissociate or hydrolyze from the microbead during orafter intravascular infusion. Preferably, the adenosine derivative iscoupled to a carboxylate modified microbead, since these derivatizedmicrobeads are readily available.

In a preferred embodiment, using carboxylate modified microbeads, themicrobeads are first contacted with an anionic/cationic exchange resinto fully remove all water-soluble ions, including polymeric materials,leaving only the surface carboxylate groups on the microbeads.Generally, the carboxylate modified microbeads are simply stirred indeionized water with an excess of the ion exchange resin. The exchangeresin: microbead w/w ratio should be about 2:1 to about 5:1. Afterremoval of the ion exchange resin, the pH of the filtered colloidalmixture can be titrated with sodium hydroxide to a pH of about 6.0-8.0,preferably about 6.5-7.5. The microbeads are then activated by reactionwith 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide at a molar ratio ofabout 5:1 in combination with N-hydroxysuccinimide at a molar ratio ofabout 4:1. The N-hydroxysuccinimide stabilizes the acylurea activatedintermediate. The activated microbead mixture which is produced is thenready for direct coupling to an adenosine derivative.

Preferred adenosine agonists which may be covalently bonded tocarboxylate modified microbeads or to dextrans include all adenosineagonists which have a free primary or secondary amino group availablefor reaction with a free carboxyl group or reactive derivative thereof(anhydride, acid halide) or with reactive isothiocyanate,N-hydroxysuccinimide ester, maleimide, or sulfonyl chloride groups. Theagonists may be bonded directly to an available carboxyl group on thepolymer microbead or, optionally, may be bonded to a free carboxyl groupof a spacer molecule, where the spacer molecule is itself bound to themicrobead.

Suitable adenosine agonists include N⁶ -phenyladenosines, N⁶ --C₅₋₈cycloalkyl adenosines N⁶ --C₁₋₆ alkyl-adenosine-5'-uronamides,2-halo-adenosines, as well as the corresponding deazaadenosinecompounds, for example N⁶ -1-methyl-2-phenethyl-1-deazaadenosine, N⁶-cyclopentyl-1-deazaadenosine, N⁶ -cyclohexyl-1-deazaadenosine.Synthetic methods for preparing suitable adenosine agonists are wellknown in the art. See Jacobson et al, Biochem. Pharm., 36(10):1697-1707(1987); Daly et al, Biochem. Pharm., 35(15):2467-2481 (1986); Cristalliet al, J. Med. Chem., 31:1179 (1988); Bridges et al, J. Med. Chem.,31:1282-1285 (1988); Jacobson et al, FEBS Letters, 225:97-102 (1987).Preferred agonists have the structure shown below: ##STR1## where R₁ isa substituent which contains a free amino group. Examples of substituentR₁ include NH₂ ; NH₂ --(CH₂)_(n) NH--, where n=1-10; NH₂ --C₆₋₂₀arylene-; NH₂ --C₅₋₆ cycloalkylene- and --NH--C₆ H₅ --CH₂ CONH--C₆ H₅--CH₂ CONH--(CH₂)_(n) --NH₂. Preferably, R₁ is amino. In the formulashown above, R₂ and R₃ may be any substituent which allows the coupledagonist to retain activity. Suitable substituents R₂ and R₃ includehydrogen, halogen, oxo (=0, R₃ only) C₁₋₆ alkyl, --NH--(CH₂)_(o) --C₆ H₅--(CH₂)_(p) --COOH, where o and p are 1-4, etc. Substituent R₄ isgenerally hydroxymethyl (CH₂ OH) but also includes C₁₋₆ alkylcarboxamido(C₁₋₆ alkyl-NHCO--) and C₃₋₆ cycloalkylcarboxamido (C₃₋₆cycloalkyl-NHCO--) groups.

Specific examples of suitable adenosine agonists include N⁶-octylaminoadenosine,2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine;adenosine; N⁶ -[[[(2-aminoethyl)amino]carbonyl]methyl]phenyl]adenosine(adenosine amine cogener, ADAC); N⁶ -benzyladenosine; CGS-21680;2-chloroadenosine; 2-chloro-N⁶ -cyclopentyladenosine; CV-1808; N⁶-cyclohexyladenosine (CHA); N⁶ -cyclopentyladenosine (CPA);5'-(N-cyclopropyl)-carboxamidoadenosine; 1-deaza-2-chloro-N⁶-cyclopentyladenosine; DPMA (PD-125944); 5'-N-ethylcarboxamidoadenosine(NECA); N⁶ -methyladenosine; α,β-methylene ATP lithium salt;5'-N-methylcarboxamidoadenosine; 1-methylisoguanosine; 2-methylthio-ATP;N⁶ -phenyladenosine; N⁶ -phenylethyladenosine;1-phenyl-2-isopropyladenosine (PIA), RR(-)-; PIA, S(+)-; N⁶-hydroxylphenylisopropyladenosine (HPIA); and N⁶-azidophenylethyladenosine (AZPNEA).

When R₁ is amino, a spacer molecule is generally used to bind theadenosine agonist to the microbead. Suitable spacer molecules include,for example, ω-aminocarboxylic acids in which the free ω-amino group isavailable to form an amide bond with the carboxylate group on themicrobead and the carboxylic acid group of the spacer is available toform an amide bond with the amino group on the adenosine agonist (R₁).These spacer groups, therefore, form stable diamide linkages covalentlylinking the adenosine agonist to the carboxylate modified microbead.Preferred ω-aminocarboxylic acids are C₃₋₁₂ alkylene, C₆₋₁₂ arylene andC₇₋₁₅ aralkylene ω-aminocarboxylic acids.

Spacer molecules suitable for reaction with microbeads having surfaceamidic, aldehydic, aromatic amine, hydrazidic or hydroxyl groups willcontain a functional group suitable for reaction with the amino group ofthe adenosine derivative, such as a carboxylic acid, acid anhydride oracid halide group, as well as a functional group suitable for reactionwith the surface functional group on the microbead. Functional groups onthe spacer molecule suitable for reaction with amide, aldehyde, aromaticamine, hydrazide or hydroxyl groups on the microbead include carboxylicacid, anhydride and acid halide groups. Amine and hydroxyl groups on thespacer molecule are suitable for reaction with surface carboxylic acidor aldehyde groups. Hydroxyl groups and amine groups may be protected ifnecessary using known acid or base stable protecting groups. Syntheticprocedures for derivatizing carboxylate modified polymer beads to formsurface amidic, haldehydic, amine, hydrazidic and hydroxyl groups, aswell as synthetic procedures to covalently couple spacer molecules tothese polymer beads are well known and any of the synthetic proceduresmay be used to couple the adenosine derivative to the microbead in thepresent invention. See, for example, Uniform Latex Particles, Seradyne,Inc., Indianapolis, Ind. (1987) and the references cited therein andJacobson et al, J. Med. Chem., 32:1043-1051 (1989).

Adenosine antagonists which can be coupled to microbeads or dextrans toprepare the compounds of the present invention in a similar mannerinclude 9-substituted adenosines, benzo[g]pteridines, xanthines,methylxanthines, such as aminophylline, etc. Other adenosine antagonistssuitable for use in the present invention include 8-phenyltheophilines,1,3-di-C¹⁻⁶ -alkyl-8-phenylxanthines, 1,3-di-C₁₋₆-alkyl-8-(p-sulfophenyl)xanthines, where the phenyl group in thesecompounds may be substituted with a halogen (Cl, Br, I), amino, COOH,SO₃ , OH or C₁₋₆ -alkyl groups.

Preferred adenosine antagonists have the structure shown below: ##STR2##where R₁ is C₁₋₆ alkyl (preferably methyl, ethyl or propyl), R₂ is C₁₋₆alkyl (preferably methyl, ethyl or propyl) and R₃ is a substituenthaving a free amino group available for bonding to the carboxylate groupof a carboxylate modified microbead. Suitable substituents R₃ includeamino, phenylamino, NH₂ CHCH₂ NH₂ --C₆ H₅ --O--CONH(CH₂)_(q) NH--(COCH₂--C₆ H₅ --)_(r) --NH₂, where q=1-6 and r is ) or 1. Methods forsynthesizing suitable adenosines antagonists are known. See for exampleR. F. Bruns, Biochem. Pharm., 30:325-333 (1981); Jacobson et al, J. Med.Chem., 32:1043-1051 (1989); Daly et al, J. Med. Chem., 28:487 (1985);Stiles et al, Molec. Pharm., 32:184-188 (1987); Jacobson et al, Molec.Pharm., 29:126-138 (1986); and Jacobson et al, Proc. Natl. Acad. Sci.USA, 83:4089 (1986).

Specific adenosine antagonists which may be used in the presentinvention include aminophylline; 1-allyl-3,7-dimethyl-8-phenylxanthine;theophylline ethylenediamine; 1-allyl-3,7-dimethyl-8-sulphoxanthine;7-(β-chloroethyl)theophylline;4-amino-N-[2-[[[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8yl)phenoxy]acetyl]amino]ethylbenzeneacetamide;8-[4-[[[[(aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine;8-cyclopentyl-1,3-dimethylxanthine; 8-cyclopentyl-1,3-dipropylxanthine;1,3-diethyl-8-phenylxanthine; 1,3-dimethylxanthine (theophylline);1,7-dimethylxanthine (paraxanthine); 3,7-dimethylxanthine (theobromine);1,3-dipropyl-7-methylxanthine; 1,3-dipropyl-8-p-sulfophenylxanthine;1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine;3,7-dimethyl-1-propargyl xanthine; PACPX;7-(β-hydroxyethyl)theophylline; 3-isobutyl-1methylxanthine;8-[4-[[[[[2-(4-aminophenylacetylamino)ethylamino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine(PAPA-XAC); 8-phenyltheophylline; 3-(n-propyl)-xanthine (enprofylline);8-(p-sulfophenyl)-theophylline; and xanthine amine cogener (XAC).

When R₃ is amino or phenylamino, a spacer molecule may be optionallyused to covalently bind the adenosine antagonist to the carboxylatemodified microbead. The spacer compounds noted above for use withadenosine agonists are also suitable for use with the adenosineantagonists in which R₃ is amino or phenylamino.

Generally, freshly activated microbeads are added to an alcohol/water ordimethylsulfoxide/water mixture and the adenosine agonist or antagonistis then charged into this mixture and stirred to allow amide bondformation. The adenosine derivative coupled to the microbead can bereadily obtained by centrifuging the resulting solution. Suitablealcohols include C₁₋₆ -alkanols, preferably ethanol or isopropanol.Generally, a slight excess of the adenosine derivative is added relativeto the surface charge equivalent of the microbead to provide high yieldsof the coupled product.

Dextrans suitable for use in the present invention are dextrans having amolecular weight ranging from 1,000 Daltons to 5,000,000 Daltons(1-5,000 kD). Suitable dextrans are commercially available (SigmaChemical Co.) having a wide range of average molecular weights. Dextranshaving a specific average molecular weight range are purified withdialysis to exclude low molecular weight contaminants. Preferred highmolecular weight dextran has a molecular weight of about1,000,000-2,000,000 Daltons (1,000-2,000 kD). Preferred low molecularweight dextrans have a molecular weight of about 1,000-10,000 Daltons(1-10 kD).

The adenosine derivative can be covalently coupled to the dextran by anyknown coupling reaction to produce a covalent bond which is stable underphysiological conditions. A preferred method of covalently bonding theadenosine derivative to the dextran is analogous to the cyanogen bromidemethod of Axen et al, Nature, 214:1302 (1967) to bind protein tooligosaccharides. In this method, a dilute aqueous solution of thedextran is reacted with cyanogen bromide to form cyanate groups whilemaintaining a basic pH through the addition of dilute sodium hydroxide.The activated dextran containing reactive cyanate groups may be used fordirect coupling to the desired adenosine derivative.

Reaction of the activated dextran with an adenosine derivative having afree amino group is believed to form a carbamate linkage between thedextran and the adenosine derivative (Axen et al). See also Armstrong etal (Biochem. Biophys. Res. Comm., 47:354-360 (1972)).

The intravascular perfusion of high molecular weight adenosine agonists(about 1,000,000 Daltons or more) and adenosine agonists bound tomicrobeads cause coronary dilation, a negative dromotropic effect anddepression of spontaneous ventricular rhythm. Since the high molecularweight compounds remain in the vascular lumen, these effects are thoughtto be the result of activation of intravascular endothelial receptors bythe adenosine agonist.

In hearts perfused at a constant coronary flow, intracoronary perfusionof microbead (microsphere) bound agonists produce coronary dilationresulting in a decrease in coronary perfusion pressure and inventricular developed pressure as shown in FIGS. 2(A) and 2(B). Theseeffects are reversible and are inhibited by the adenosine receptorblocker 8-sulphophenyltheophylline (SPT). The mean decrease in coronaryperfusion pressure (control =49 ±1.7 mmHg) and ventricular developedpressure (control =65 ±3.5 mmHg) from several experiments with microbead(microsphere) bound NOA (SPH-NOA) and microbead (microsphere) bound ADAC(SPH-ADAC) are shown in FIGS. 2(A) and 2(B). These changes werestatistically significant with a value of p<0.05. Perfusion with controlmicrobeads did not exhibit these effects.

Perfusion with microbead-bound agonists also causes a negativedromotropic effect, These compounds cause a shift upward and to the leftof atrial-ventricular (A-V) delay-atrial frequency curves as shown inFIGS. 3(A) and 4(A). At any frequency of atrial pacing, the A-V delaywas lengthened and the maximal frequency of atrial pacing to yield a 1:1A-V transmission was reduced. These effects were inhibited by SPT asshown in FIG. 3(A). FIGS. 3(B) and 4(B) plot the differences betweenexperimental values and corresponding control values of A-V delays atvarious frequencies of atrial pacing. The mean values of severalexperiments were statistically significant for all points at a p ≦0.02.The differences between control maximal frequency of atrial pacing for a1:1 A-V transmission (mean=5.1 ±0.04 Hz) minus the correspondingexperimental maximal frequencies for SPH-NOA and SPH-ADAC are shown inFIG. 2(C). All values have a statistical significance of p≦0.01.Perfusion with control microbeads did not exhibit these effects.

Infusion of the agonists of the present invention also effects thespontaneous ventricular rhythm and glycolytic flux. Intracoronaryinfusion of SPH-NOA caused the rate of spontaneous ventricular rhythm todrop from a control value of 149±9 to 116±2 beads/min (p≦0.01). Upontermination of SPH-NOA infusion, spontaneous ventricular dischargerecovered to the initial control value. FIG. 2(D) shows thatintracoronary infusion of SPH-NOA causes a significant reduction inglycolytic flux (p≦0.05). Perfusion with control microbeads did notaffect spontaneous ventricular rhythm of glycolytic flux.

Bolus injections of adenosine allow one to assess the ability of theantagonist derivatives of the present invention to block adenosine A₁receptors. FIG. 6(A) shows the rapid drop in mean coronary pressurecaused by administration of a 10.0 μl adenosine bolus (10³ M) withsubsequent recovery. The vascular response was resistant to bolusinjections of adenosine (p<0.001) during infusion of SPH-XAC (6.0 mg/ml)as shown in FIG. 6(B). With an equivalent molar amount of free XAC (10⁻⁷M), the vascular response to adenosine bolus injection was not blockedas shown in FIG. 6(C). The vascular response was affected only in thatthe magnitude of the coronary pressure drop was greater (p<0.05) due toa higher baseline mean coronary pressure observed during free XACinfusion. Only at concentrations of 10⁻⁵ M free XAC, was there acomplete block of the mean coronary pressure drop with adenosine bolusinjections. See FIG. 6(D), p<0.0001. The 100 fold difference inpharmacological effect between microbead bound XAC and free XAC isillustrated in FIG. 6(E) where infusion of 0.06 mg/ml microbead-XAC didnot block the vascular effects of adenosine bolus injection.

There is no pharmacological difference in dromotropic effects betweenmicrobead-XAC and free-XAC. See FIGS. 7(A) 7(B), where equivalent molaramounts of microbead-XAC and free-XAC both block the negativedromotropic effects of adenosine bolus injections.

FIGS. 8 and 9 show the dromotropic and vascular effects of themicrobead-bound antagonists of the present invention under hypoxicconditions. FIGS. 8(A) and 8(B) show the dromotropic effects of a 2.0minute period of perfusion with K-H equilibrated with 95% N₂ +5% CO₂where the maximal prolongation of the A-V interval is reduced by greaterthan 50% (p <0.01) during infusion of equal molar amounts of free andmicrobead-XAC.

The vasodilatory effects of endogenous adenosine were studied under twovarying degrees of hypoxia (0% O₂ and 20% O₂) in order to assess whetherthe degree of hypoxia would reveal any differential effect between equalmolar amounts of microbead-XAC and free-XAC. FIG. 9 shows that during anindividual experiment (FIGS. 9(A-C)) the mean coronary pressure dropdecreased by a similar magnitude whether microbead-XAC or free-XAC wasinfused. Neither compound significantly blocks the vasodilatory effectsof hypoxia. There is no statistical difference between the control andexperimental conditions during maximal levels of hypoxia as shown inFIG. 9(D) at 0% O₂ or minimal levels of hypoxia as shown in FIG. 9(E) at20% O₂.

The adenosine derivatives covalently coupled to microbeads of thepresent invention have the advantage that these particles are confinedsolely to the intravascular compartment. However, the colloidal natureof these microbead-bound pharmaceuticals results in a low effectiveconcentration due to the insolubility of the particles in the aqueousblood system. The dextran-coupled adenosine derivatives of the presentinvention are medium and large water soluble compounds, however, andhave a substantially higher effective concentration.

As with microbead-bound adenosine derivatives, the dextran-boundderivatives also cause a decrease in coronary perfusion pressure and inventricular developed pressure. The results of an individual experimentwith dextran-ADAC are shown in FIG. 10. Similar results are obtainedwith dextran-NOA perfusion.

Sustained intracoronary infusion of dextran-ADAC causes a gradual risein the A-V delay (FIG. 11) similar to A-V delay results observed withmicrobead-ADAC.

FIG. 11 shows the anti-adrenergic effect of dextran-ADAC. Duringperfusion of dextran ADAC (0.1 mg/ml), a bolus of isoproterenol (0.0001μg) was administered. The positive dromotropic effects of isoproterenolwere considerably depressed and shorter than when isoproterenol wasgiven alone. See the lower trace in FIG. 11. Termination of thedextran-ADAC infusion reversed the negative dromotropic effects.

As noted above, hypoxia causes an increase in the A-V delay which hasbeen attributed to a rise in the interstitial levels of adenosine, whichact directly on A-V nodal cells depressing the generation of theiraction potentials. This effective adenosine is blocked bymethylxanthines. The negative dromotropic effects of hypoxia are blockedby 0.1 mg/ml dextran-XAC (FIG. 12).

Adenosine antagonists coupled to high molecular weight dextrans ormicrobeads have only minor coronary and dromotropic effects whenadministered intracoronarily. These adenosine antagonists (at 0.01 to 10mg/ml) block hypoxia-induced lengthening of the auricular-ventricularinterval without affecting the associated coronary dilation. Using highmolecular weight compounds, therefore, one is able to cause prolongedcoronary dilation, a negative dromotropic effect and depress spontaneousventricular rhythm in mammals.

The high molecular weight adenosine agonists of the present inventionare useful in treating hypertension, cardiac arrhythmias and inpreserving myocardial tissue. Infusion of high molecular weight agonists(at 0.01 to 10 mg/ml) results in coronary dilation and a drop inintravascular pressure (blood pressure), offering a selective treatmentfor hypertension. The negative dromotropic effect of the high molecularweight agonists (at 0.1 to 10 mg/ml) can be used to treattachycardiarrhythmias, such as supraventricular tachycardia. Infusion ofthe high molecular weight adenosine agonist increases the A-V intervalthereby offering effective treatment for tachycardia.

Additionally, the adenosine agonists (at 0.1 to 20 mg/ml) of the presentinvention are useful in reducing the size of a myocardial infarction inthe same manner in which adenosine itself is used to reduce infarct sizeand improve regional ventricular function in ischemic zones of theheart. See Olafsson et al, Babbit et al and Liu et al.

The covalently coupled adenosine agonists and antagonists of the presentinvention may be administered by intracoronary infusion at anintracoronary infusion concentration of about 0.01 mg/ml to about 20mg/ml, preferably about 0.05-0.50 mg/ml. The solutions or suspensions ofcovalently coupled adenosine agonists and antagonists are preferablycontinuously infused.

Pharmaceutical compositions containing the covalently coupled adenosinederivatives are also within the scope of the present invention. Thepharmaceutical compositions include solutions or suspensions of thecovalently coupled adenosine agonist or antagonist in a sterile salineor buffer solution suitable for infusion into the patients vascularsystem. Solutions or suspensions in sterile 5% saline,dextrose-5%-saline or phosphate buffered saline solutions having aconcentration of about 0.01 mg/ml to about 20 mg/ml, preferably about0.05-0.50 mg/ml of the covalently coupled adenosine agonist and/orantagonist are preferred. These pharmaceutical compositions may beadministered repeatedly by intravenous injection or continuously by slowintravenous infusion.

Other features of the invention will become apparent during the courseof the following descriptions of exemplary embodiments which are givenfor illustration of the invention and are not intended to be limitingthereof.

EXAMPLES I. Microbead Coupling

Activation of microbeads. Carboxylate modified beads with a surfacecharge density of 0.3 meq/g and mean diameter of 0.07 μm (Seradyn Inc.)were prepared by diluting to less than 5% solids with deionized water.The solution was stirred for 2 hours with a 50/50 anionic/cationicexchange resin at a 2:1 wt/wt ratio of resin to beads. Resin wasremoved, thereafter, the pH of the filtered colloidal mixture wastitrated with 1.0M NaOH to a value of 7.5. The microbeads were thenactivated at 4° C. with 1-(3-dimethyl-aminopropyl)-3-ethyl carbodiimidein a 5:1 mole ratio followed by the addition of N-hydroxysuccinimide ina 4:1 mole ratio in order to stabilize the O-acylurea activatedintermediate. This mixture was then ready for direct coupling.

N⁶ -octylamine adenosine (NOA) conjugation to activated microbeads. Tofreshly activated microbeads was added isopropyl alcohol and deionizedwater to create a 50/50 mixture of 15 ml with 100 mg beads per totalvolume mixture. This solution was cooled to 4° C. and charged with NOAat a 1.5/1.0 mol ratio of adenosine agonist to surface chargeequivalents. The solution was vortexed at 5 minute intervals for 20minutes and allowed to stand at 4° C. overnight. The reaction mixturewas then centrifuged at 40,000×g for three hours, and the supernatantwas decanted and resuspended in deionized water with a microtipsonifier. This purification procedure was repeated 5 times. Prior tocoronary infusion, these spheres were resuspended in deionized water ata concentration of 5-6 mg/ml.

N⁶ -[4-(2-Aminoethylaminocarbonylmethyl)phenyl]adenosine (ADAC)conjugation to activated microspheres. The activated microspheresuspension was diluted with DMSO and deionized water to create a 70/30v/v mixture respectively at a total volume of 30 ml/100 mg spheres. Aconcentrated stock solution of ADAC was prepared in a DMSO deionizedwater mixture (70/30 v/v respectively) and this ADAC solution was addedto the microsphere suspension a 1.5/1.0 mol ratio of ADAC to surfacecharge equivalents. The reaction time was 2 h at 4° C. The reactionmixture was then dialyzed for 10 h at 4° C. against deionized waterthrough a dialysis membrane with a cutoff at 14,000-16,000. Thispurification step was repeated four times. In order to concentrate thespheres prior to its use, after the last dialysis step the spheresuspensions were centrifuged at 40,000×g for 3 h, the fluid was decantedand spheres resuspended in deionized water at a concentration of 5-6mg/ml. In all experiments microbeads were intracoronarily infused at aconcentration of 0.05 mg/ml.

In order to test for the presence of free agonist in the sphere-agonistsuspension, an aliquot of this suspension was filtered through acentrifugal ultrafiltration unit with a cutoff of 30,000 kD (MilliporeULTRAFREE-20 filter unit, 10,000 NMWL). This step retained themicrobeads particles in the filter and resulted in a microbead freefiltrate that could only contain free agonist.

8-[4-(2-aminoethylaminocarbonyl methoxy)phenyl]-1,3-dipropylxanthine(XAC) conjugation to activated microbeads. 5.0 mM XAC (RBI) solutionswere prepared in 0.1N NaOH and 1.0% NaCl, and subsequently was titratedto pH 9.2 with 0.1M HCl prior to use. This XAC solution was then addedto the microbead suspension in a 1.0/1.0 mole ratio of XAC tomicrobead-surface charge equivalents. The reaction time was carried outovernight at 4° C. The reaction mixture was then dialyzed for 48 hoursat 4° C. against deionized water through a dialysis membrane with amolecular weight cutoff at 14,000-16,000. The bead-XAC solution was thencentrifuged at 10,000×g for 10 min. in order to remove excessprecipitate and was followed by 6 successive centrifuge washings at40,000×g in order to remove low molecular weight contaminants. In thecase of the washings, the pelleted microbeads were each time resuspendedin deionized H₂ O with a microtip sonifier. In all experimentsmicrobeads were infused at a concentration of 6.0 mg/ml, and as themicrobeads were infused at a rate of 0.1 ml/min. and diluted withperfusion media at a rate 10.0 ml/min., the final intracoronaryconcentration was 0.06 mg/ml. In all further reference to microbeadconcentration, it is the infusion concentration which is expressed.

The efficiency of the conjugation reaction was determined byspectrophotometric means and by radioisotope spiking of reaction mediawith ³ H labeled XAC (NEN Research Products) followed by scintillationcounting of the purified reaction product. For the spectrophotometricquantification of covalently bound XAC it was necessary to dissolve themicrobead conjugates in pyridine in order to eliminate the scatteringeffects of the microbeads.

In order to test for the presence of "free" antagonist in thebead-antagonist suspension, an aliquot of this suspension was filteredthrough a centrifugal ultrafiltration unit with a cutoff of 30 kD(Millipore ULTRAFREE-20 filter unit, 10,000 NMWL). This step retainedthe microbead particles in the filter and resulted in a microbead freefiltrate that could only contain "free" antagonist.

Two types of control beads were prepared either omitting: a)1-(3-dimethylaminopropyl)-3-ethyl carbodiimide during the activationstep or b) the conjugation moiety. In all experiments microbeads wereintracoronarily infused at a concentration of 0.06 mg/ml.

II. Dextran Coupling

Activation of Dextran. The cyanogen bromide method was used for alldextran conjugation reactions. The dextran was purified by dialysis(dialysis membrane cutoff 16 kD) to exclude small molecular weightcontaminants. Subsequently a stirred solution of 0.01% dextran wascharged with cyanogen bromide in 3 equal portions at 15 min. intervals,to yield a final 50 wt/wt % of cyanogen bromide to dextran solution. ThepH of this activation step was monitored constantly and maintained at pH10.7 by addition of 1M NaOH. Thirty min. after the addition of the lastcyanogen bromide portion, the solution was used for direct coupling.

N⁶ -[4-(aminoethylamino)carbonylmethylphenyl]adenosine (ADAC)conjugation to activated dextran. Reaction with ADAC proceeded as abovefor direct coupling of ADAC to activated microbeads, except ADAC wasadded at a 50 wt/wt % to dextran, and the ADAC was solubilized in 0.1Macetic acid. Coupling time and purification were same as above exceptthat final dialysis was against 0.9% saline solution.

XAC conjugation to activated dextran. XAC was solubilized in 0.1M NaOHand coupled to activated dextran according to the procedure disclosedabove.

III. Microbead Experiments

Isolated saline perfused hearts. Male guinea pig (350-400 g) wereanesthetized with an intraperitoneal injection of ketamine/xylazine(80/20 mg/Kg body weight) and heparin (500 U). The heart was removed andretrogradely perfused via a non-recirculating perfusion system atconstant flow. Perfusion was initiated at a rate of 25.0 ml/min for 5.0minutes and followed by 25.0 min equilibration period of perfusion at arate of 10.0 ml/min. All experimental measurements were initiated afterthis period of equilibration. The perfusion media was Krebs-Henseleitsolution (K-H) with the following composition (mM): NaCl 117.8, KCl 6,CaCl₂ 1.6, NaHCO₃ 25, NaH₂ PO₄ 1.2, NaEDTA 0.0027, and glucose 5.0. Thissolution was equilibrated with 95% 0₂, 5% CO₂ at 37° C. and had a pH of7.4. Subsequent studies during induced hypoxia used an equilibratedperfusion media with either (95% N₂ +5% CO₂) or (75% N₂ +20% O₂ +5%CO₂).

All experiments were performed at a constant coronary flow of 10.0ml/min and the coronary perfusing pressure was recorded continuously viaa side arm of the perfusing cannula and had a control value of 48±2.5mmHg.

One pair of stimulating electrodes was placed in the apex of the rightatria and electric square pulses of 2.0 msec duration and two timesthreshold were applied. To record the electrocardiogram one electrodewas placed in the right atria and a second electrode in the leftventricle. These two electrodes were connected to an oscilloscopesynchronized with the atrial pacing stimulator while theauricular-ventricular delay (A-V delay; msec) was continuously monitoredand measured as the time interval between the application of thestimulus to the atria and the initiation of the rising phase of theventricular electrical signal. The time between the application of thestimulus and the atrial electrogram remained constant (18±1.3 msec)throughout all the experimental manipulations.

Measurements of A-V delay during adenosine bolus experiments wereperformed with the aid of a polaroid camera mounted on the oscilloscopedisplay panel. Manual operation of the shutter speed was sufficient tocapture A-V delay prior to the gradual development of complete heartblock (Mobitz type 11); so that each filmed exposure containedsuccessive electrocardiographic tracings where each beat (occurring at atime equivalent to 1/[stimulation frequency]) showed the gradualprolongation A-V delay to complete heart block.

Studies during hypoxia with microbead-XAC. The effect of hypoxia oncoronary pressure and A-V delay were studied under control conditionsand during constant infusion of "unbound" XAC and "microbead-bound"(bead-XAC) XAC. These studies were performed at two levels of reducedoxygen tension. For control experiments the K-H solution wasequilibrated with 95% O₂ +5% CO₂ and subsequent hypoxic studies with 95%N₂ +5% CO₂ or 75% N₂ +20% O₂ +5% CO₂. In all cases hypoxic conditionswere initiated, after 25 min. of equilibration during controlconditions, by rapid-manual switching to a parallel perfusion systemequilibrated with the appropriate gas mixture. Hypoxic conditions weremaintained for 2.0 minutes in all experiments. Thereafter, coronarypressure and A-V delay were continuously monitored as a function oftime. A structural dead space in our perfusion apparatus, ofapproximately 2.0 ml, was responsible for the aberrant cardiovasculareffects seen in the initial one minute during hypoxia.

Electronmicroscopy. Electronmicroscopy studies were conducted todemonstrate that the 0.07 μm diameter microbeads when infusedintracoronarily remained confined to the intravascular space. Asdescribed above, the hearts were isolated and perfused with K-H at arate of 10.0 ml/min and infused with the microbeads (6.0 mg/min ) at arate of 0.1 ml/min. for 5.0 min. Thereafter, while the infusion ofmicrobeads was sustained, the perfusion with the K-H solution waschanged to one of glutaraldehyde (50 mM phosphate buffer, pH 7.4,glutaraldehyde 2.5%) and perfused at the same rate for a period of 10.0min. The heart was removed and the free wall of the left ventricle wasminced into small cubes of 1.0 MM3 and left overnight in theglutaraldehyde solution. The ventricular tissue was then rinsed inphosphate buffer and post fixed for 60.0 min in 1% OsO₄ solutionfollowed by a water rinse. Following alcohol dehydration, the tissueswere embedded in epon resin and sections of 0.6 μm to 0.9 μm were cutand stained with lead citrate and uranyl acetate. Sections were thenviewed and photographed in a transmission electronmicroscope.

The electronmicroscopic studies showed that no microbeads could beobserved in the myocardial parenchymal tissue after 5.0 min. ofmicrobead infusion.

Bioassay to detect the presence of free antagonist in venous effluentsFor these studies two heart preparations were utilized; a "donor" heartfrom which venous effluent was collected and a "recipient" heart fromwhich venous effluent was assayed. Accordingly, as an index forevaluating the possibility of "hydrolyzed" XAC from bead-XAC conjugatesduring infusion into the "donor" heart, the dromotropic effects ofadenosine bolus injections were evaluated in the "recipient" heartduring perfusion with donor effluent. Specifically, a control venouseffluent aliquot of 1 00 ml from the "donor" heart was collected priorthe coronary infusion of the bead-XAC into the donor heart. Thereafter,in order to determine if the bead-XAC complex was hydrolyzed into freeXAC and beads, during its passage through the heart, microbeads wereperfused into the "donor" heart and experimental venous effluents weresimultaneously collected for about 20 minutes. The experimental venouseffluents were divided into two equal volume aliquots. In one of thesealiquots the bead-agonist complexes were removed by filtering it firstthrough a membrane of 0.24 μm pore size, thereafter, the filtrate waspassed through a centrifugal ultrafiltration unit with a cutoff of 30kD. This step retained the microbeads particles in the filter andresulted in a microbead free filtrate that could contain only freeagonist. The other venous effluent aliquot was assayed with bead-XACremaining in solution. The different venous effluents from the donorheart were equilibrated with 95% O₂ +5% CO₂ and brought to 37° C. priortheir infusion into the "recipient" heart and they were assayed in thefollowing order; control effluent, experimental effluent with removedmicrobeads and experimental effluent with microbeads. The results showthat control and experimental effluents with removed microbeads have noeffect in the recipient heart while the experimental effluents withmicrobeads have the same effects in the recipient heart as in the donor.These results establish the stability of the chemical bond betweenagonist/antagonist and the microbeads.

Measurements of coronary resistance. Coronary resistance was determinedfrom the ratio of coronary perfusion pressure to coronary flow. In allthese experiments coronary flow was maintained constant at 10 ml/min.

Measurements of ventricular contraction (Myc.). Via the left atria afluid filled balloon was introduced into the left ventricle. Diastolicpressure was adjusted to about 10 mmHg and the developed pressure (Myc,mmHg) continuously determined.

Studies on A-V. A-V delay was determined at various frequencies ofatrial stimulation and A-V delay (ordinates)-frequency (abscissas) plotswere generated.

Studies on spontaneous ventricular rhythm, (V_(t)). Spontaneousventricular rhythm was induced by destroying the A-V nodal area. Forthis purpose, a large incision was made in the right atria to clearlyexpose the coronary sinus ostium, the ventricular septum and thetricuspid valve. Destruction of the A-V nodal area was achieved bycrushing with small forceps the superficial tissue laying between theostium and cardiac valves. This manipulation resulted in blockade of theimpulses from atria to ventricle and appearance of spontaneousventricular rhythm (V_(t), beats/mis).

Measurements of glycolytic flux. To determine glycolytic flux aperfusion media containing 0.1 μCi/ml [D-3-³ H]glucose (15 Ci/mmol;American radiolabeled Chemicals Inc.) was perfused and after 15 min ofequilibration in this medium, coronary venous effluents was continuouslycollected successively every 2 min throughout the duration of theexperiment. At the end of the experiment the heart was removed, largevessels dissected out and placed in an oven to dry and thereafterweighed. Glycolytic flux was determined from analysis of ³ H₂ O contentin the venous effluent aliquotes since production of ³ H₂ O during theenolase reaction of the Embden-Meyerhoff pathways is directlyproportional to the flux in the glycolytic pathway To separate ³ H₂ Ofrom [³ H]glucose, 1 ml sample of perfusate was placed on a 1×4 cmcolumn of Dowex-1 borate which retained labeled glucose. The columneffluents was collected directly into counting vial and the columnfurther washed with 2 ml of water. To these 3 ml of effluentscintillation liquid was added and counted. The rate of ³ H₂ O releasewas determined as cpm/min per g dry weight and used to calculateglycolytic flux from the specific radioactivity of the perfused glucose.Glycolytic flux was expressed as μmol/min per g dry weight.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United State is:
 1. An adenosine agonist or antagonist having apurine ring structure covalently coupled to a water-soluble dextran,wherein said dextran is covalently coupled to the 6- position or to the8- position of said purine ring structure by reaction of cyanogenbromide activated dextran with the free amino group of a linker which iscovalently bonded to said 6- position or 8- position and said linker isselected from the group consisting of NH₂ --(CH₂)_(n) --NH-- wheren=1-10, NH₂ --C₆₋₂₀ arylene-, NH₂ --C₅₋₆ cycloalkylene-, --NH--C₆ H₅--CH₂ CONH--C₆ H₅ --CH₂ CONH --(CH₂)_(n) --NH₂, --C₆ H₅ --NH₂,--CH(NH₂)CH₂ NH₂ and --C₆ H₅ --O --CH₂ --CONH(CH₂)_(q) NH --(COCH₂ --C₆H₅ --)_(r) --NH₂ where q=1-6 and r=0-1.
 2. The covalently coupledadenosine agonist or antagonist of claim 1, wherein said dextran has amolecular weight of 1 kD-5000 kD molecular weight.
 3. The covalentlycoupled adenosine agonist or antagonist of claim 1, wherein said dextranhas a molecular weight of 1,000-2,000 kD.
 4. The covalently coupledadenosine agonist or antagonist of claim 1, wherein said dextran has amolecular weight of 1-10 kD.
 5. The covalently coupled adenosine agonistof claim 1, wherein said adenosine agonist or antagonist is adenosine.6. The covalently coupled adenosine agonist or antagonist of claim 1,wherein said covalently coupled agonist or antagonist selectively bindsadenosine A₁ receptors.
 7. The covalently coupled adenosine agonist orantagonist of claim 1, wherein said covalently coupled agonist orantagonist selectively binds adenosine A₂ receptors.
 8. The covalentlycoupled adenosine agonist or antagonist of claim 1, wherein saidadenosine agonist or antagonist is an adenosine agonist.
 9. Thecovalently coupled adenosine agonist or antagonist of claim 1, whereinsaid adenosine agonist or antagonist is an adenosine antagonist.
 10. Thecovalently coupled adenosine agonist of claim 8, wherein said adenosineagonist has the structure shown below: ##STR3## wherein R₁ is NH₂--(CH₂)_(n) --NH--, NH₂ --C₆₋₂₀ arylene-, NH₂ --C₅₋₆ cycloalkylene-, or--NH--C₆ H₅ --CH₂ CONH--C₆ H₅ --CONH--(CH₂)_(n) --NH₂ , where n=1-10; R₂is hydrogen, halogen, or C₁₋₆ alkyl; R₃ is hydrogen, halogen, C₁₋₆alkyl, or oxo and R₄ is hydroxymethyl, C₁₋₆ alkyl-carboximido or C₃₋₆cycloalkyl-carboximido.
 11. The covalently coupled adenosine agonist ofclaim 8, wherein said adenosine agonist is selected from the groupconsisting of N⁶ -phenyl-adenosine, N⁶ -C₅₋₈ cycloalkyl-adenosine, N⁶-C₁₋₆ alkyl-adenosine-5'-uronamide, and 2-halo-adenosine.
 12. Thecovalently coupled adenosine agonist of claim 8, wherein said adenosineagonist is selected from the group consisting of N⁶-1-methyl-2-phenethyl-1-deazaadenosine, N⁶-cyclopentyl-1-deazaadenosine, N⁶ -cyclohexyl-1-deazaadenosine, N⁶-octylaminoadenosine,2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxylamidoadenosine,adenosine, N⁶ -(4-(2-aminoethylaminocarbonylmethylphenyl)adenosine, N⁶-benzyladenosine, 2-chloroadenosine, 2-chloro-N⁶ -cyclopentyladenosine,N⁶ -cyclohexyl adenosine, N⁶ -cyclopentyladenosine,5'-(N-cyclopropyl)-carboxamidoadenosine, 1-deaza-2-chloro-N⁶-cyclopentyladenosine, 5'-N-ethylcarboxamidoadenosine, N⁶-methyladenosine, αβ-methylene ATP lithium salt, 2-methylthio-ATP,1-methylisoguanosine, 5'-N-methylcarboxamidoadenosine, N⁶-phenyladenosine, N⁶ -phenylethyladenosine,1-phenyl-2-isopropyladenosine, N⁶ -hydroxylphenylisopropyladenosine andN⁶ -azidophenylethyladenosine.
 13. The covalently coupled adenosineantagonist of claim 9, wherein said adenosine antagonist is selectedfrom the group consisting of adenosine, xanthine and theophylline. 14.The covalently coupled adenosine antagonist of claim 9, wherein saidadenosine antagonist has the structure shown below ##STR4## wherein R₁is C₁₋₆ alkyl, R₂ is C₁₋₆ alkyl and R₃ is phenylamino, --NH₂ CHCH₂ NH₂,--C₆ H₅ --O--CH₂ --CONH(CH₂)_(q) NH --(COCH₂ --C₆ H₅ --)_(r) --NH₂,where q =1-6 and r is 0 or
 1. 15. The covalently coupled adenosineantagonist of claim 8, wherein said adenosine antagonist is selectedfrom the group consisting of aminophylline;1-allyl-3,7-dimethyl-8phenylxanthine; theophylline ethylenediamine;1-allyl-3,7-dimethyl-8-sulphoxanthine; 7- (β-chloroethyl) theophylline;4-amino-N-[2-[[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetyl]amino]ethyl benzeneacetamide; 8-[4-[[[(aminoethyl)amino]carbonyl]methyl]oxy]phenyl-1,3-dipropylxanthine;8-cyclopentyl-1,3-dimethylxanthine; 8-cyclopentyl-1,3-dipropylxanthine;1,3-diethyl-8phenylxanthine; 1,3-dimethylxanthine; 1,7-dimethylxanthine;3,7-dimethylxanthine; 1,3-dipropyl-7-methylxanthine;1,3-dipropyl-8-p-sulfophenylxanthine; 1,3-dipropyl-8-(2-amino -4-chlorophenyl)-xanthine: 3,7-dimethyl-1-propargyl xanthine; PACPX; 7-(β-hydroxyethyl) theophylline; 3-isobutyl-1-methylxanthine;8-[4-[[[2-(4-aminophenylacetylamino)ethylamino]carbonyl]methyl]oxy]phenyl-1,3-dipropylxanthine;8-phenyltheophylline; 3-(n-propyl)-xanthine;8-(p-sulfophenyl)-theophylline; and 8-[4-(2-aminoethylaminocarbonylmethoxy) phenyl]-1,3-dipropylxanthine.
 16. A pharmaceutical compositioncomprising the covalently coupled adenosine agonist or antagonist ofclaim 1 and a pharmaceutically acceptable carrier or diluent.